1. Field of Invention
This invention relates to molecules useful as agonists of the insulin-like growth factors (IGFs). More particularly, these molecules inhibit the interaction of an IGF with one or more of its IGF binding proteins. Such molecules can be used, for example, in any methods where the IGFs are used, for example, in treating hyperglycemic, obesity-related, neurological, cardiac, renal, immunologic, and anabolic disorders.
2. Description of Background and Related Art
There is a large body of literature on the actions and activities of IGFs (IGF-I, IGF-II, and IGF variants). Human IGF-I is a 7649-dalton polypeptide with a pI of 8.4 (Rinderknecht and Humbel, Proc. Natl. Acad. Sci. USA, 73: 2365 (1976); Rinderknecht and Humbel, J. Biol. Chem., 253: 2769 (1978)) belonging to a family of somatomedins with insulin-like and mitogenic biological activities that modulate the action of growth hormone (GH) (Van Wyk et al., Recent Prog. Horm. Res., 30: 259 (1974); Binoux, Ann. Endocrinol., 41: 157 (1980); Clemmons and Van Wyk, Handbook Exp. Pharmacol., 57: 161 (1981); Baxter, Adv. Clin. Chem., 25: 49 (1986); U.S. Pat. No. 4,988,675; WO 91/03253; WO 93/23071).
Like GH, IGF-I is a potent anaholic protein. See Tanner et al., Acta Endocrinol. 84: 681-696 (1977); Uthne et al., J. Clin. Endocrinol. Metab., 3: 548-554 (1974). See also Ross et al., Intensive Care Med., 19 Suppl. 2: S54-57 (1993), which is a review of the role of insulin, GH, and IGF-I as anabolic agents in the critically ill. IGF-I has hypoglycemic effects similar to those of insulin, but also promotes positive nitrogen balance (Underwood et al., Hormone Res., 24: 166 (1986); Guler et al., N. Engl. J. Med., 317: 137 (1987)). Due to this range of activities, lGF-I is being tested in humans for such widely disparate uses as wound healing, treatment of diabetes, reversal of whole body catabolic states, treatment of heart conditions such as congestive heart failure, and treatment of neurological disorders (Guler et al., Proc. Nati. Acad. Sci. USA 85: 4889-4893 (1988); Duerr et al., J. Clin. Invest., 95: 619-627 (1995); and Barinaga, Science, 264: 772-774 (1994)).
U.S. Pat. Nos. 5,273,961; 5,466,670; 5,126,324; 5,187,151; 5,202,119; 5,374,620; 5,106,832; 4,988,675; 5,106,832; 5,068,224; 5,093,317; 5,569,648; and 4,876,242; WO 92/11865; WO 96/01124; WO 91/03253; WO 93/25219; WO 93/08826; and WO 94/16722 disclose various methods of treating mammals, especially human patients, using IGF-I. In addition, clinical uses of IGF-I are described, for example, in Bondy, Ann Intern. Med., 120: 593-601 (1994).
As one specific use, IGF-I has been found to exert a variety of actions in the kidney (Hammerman and Miller, Am. J. Physiol., 265: F1-F14 (1993)). It has been recognized for decades that the increase in kidney size observed in patients with acromegaly is accompanied by a significant enhancement of glomerular filtration rate (O""Shea and Layish, J. Am. Soc. Nephrol., 3: 157-161 (1992)). U.S. Pat. No. 5,273,961 discloses a method for prophylactic treatment of mammals at risk for acute renal failure. In humans IGF-I has been shown to preserve renal function post-operatively (Franklin et al., Am. J. Physiol., 272: F257-F259 (1997)). Infusion of the peptide in humans with normal renal function increases glomerular filtration rate and renal plasma flow (Guler et al., Acta Endocrinol., 121: 101-106 (1989); Guler et al., Proc. Natl. Acad. Sci. USA, 86: 2868-2872 (1989); Hirschberg et al., Kidney Int., 43: 387-397 (1993); U.S. Pat. No. 5,106,832). Further, humans with moderately reduced renal function respond to ru short-term (four days) IGF-I administration by increasing their rates of glomerular filtration and renal plasma flow. Hence, IGF-I is a potential therapeutic agent in the setting of chronic renal failure (O""Shea et al., Am. J. Physiol., 264: F917-F922 (1993)). Despite the fact that IGF-I can enhance renal function for those experiencing end-stage chronic renal failure, the enhancements of the glomerular filtration rate and renal plasma flow induced by IGF-I short-term do not persist during long-term administration and incidence of side-effects is high (Miller et al., Kidney International, 46: 201-207 (1994)).
For complete reviews of the effect of IGF-I on the kidney, see, e.g., Hammerman and Miller, Am. J. Physiol., 265: F1-F14 (1993) and Hammerman and Miller, J. Am. Soc. Nephrol., 5: 1-11 (1994).
As to anabolic indications for IGF-I, in HIV-infected patients treated consecutively with IGF-I, the IGF-I promoted anabolism, but tachyphylaxis developed rapidly in the patients (Lieberman et al., U.S. Endocrine Meeting, June 1993 (Abst. 1664); Lieberman et al., J. Clin. Endo. Metab., 78: 404-410 (1994)). In patients with severe head injuries, a condition associated with profound hypercatabolism and nitrogen loss, infusion of IGF-I produced only a transient positive nitrogen balance. In the first week the patients experienced a positive nitrogen balance, but during the second week, a negative nitrogen balance developed (Chen et al., U.S. Endocrine Meeting, June 1993 (Abst. 1596)).
IGF-I has hypoglycemic effects in humans similar to those of insulin when administered by intravenous bolus injection (Underwood et al., Hormone Research, 24: 166 (1986)). IGF-I is known to exert glucose-lowering effects in both normal (Guler et al., N. Engl. J. Med., supra) and diabetic individuals (Schoenle et al., Diabetologia, 34: 675-679 (1991); Zenobi et al., J. Clin. Invest., 90: 2234-2241 (1992); Sherwin et al., Hormone Research, 41 (Suppl. 2): 97-101 (1994); Takano et al., Endocrinol. Japan, 37: 309-317 (1990); Guler et al., Acta Paediatr. Scand. (Suppl.), 367: 52-54 (1990)), with a time course described as resembling regular insulin. See also Kerr et al., xe2x80x9cEffect of Insulin-like Growth Factor 1 on the responses to and recognition of hypoglycemia,xe2x80x9d American Diabetes Association (ADA), 52nd Annual Meeting, San Antonio, Tex., Jun. 20-23, 1992, which reported an increased hypoglycemia awareness following recombinant human IGF-I (rhIGF-I) administration. In addition, single administration of rhIGF-I reduces overnight GH levels and insulin requirements in adolescents with IDDM (Cheetham et al., Clin. Endocrinol., 40: 515-555 (1994); Cheetham et al., Diabetolopia, 36: 678-681 (1993)).
The administration of rhIGF-I to Type II diabetics, as reported by Schalch el al., J. Clin. Endo. Metab., 77: 1563-1568 (1993), demonstrated a fall in both serum insulin as well as a paralleled decrease in C peptide levels. This indicated a reduction in pancreatic insulin secretion after five days of IGF-I treatment. This effect has been independently confirmed by Froesch el al., Horm. Res., 42: 66-71 (1994). In vivo studies in normal rats also illustrate that IGF-I infusion inhibits pancreatic insulin release (Fumsinn et at., Endocrinology, 135: 2144-2149 (1994)). In addition, in pancreas perfusion preparations, IGF-I also suppressed insulin secretion (Leahy et al., Endocrinology, 126: 1593-1598 (1990)). Despite these clear in vivo inhibitory effects of IGF-I on insulin secretion in humans and animals, in vitro studies have not yielded such uniform results.
RhIGF-I has the ability to improve insulin sensitivity. For example, rhIGF-I (70 xcexcg/kg bid) improved insulin sensitivity in non-diabetic, insulin-resistant patients with myotonic dystrophy (Vlachopapadopoulou et al., J. Clin. Endo. Metab., 80: 3715-3723 (1995)). Saad et al., Diabetologia, 37: Abstract 40 (1994) reported dose-dependent improvements in insulin sensitivity in adults with obesity and impaired glucose tolerance following 15 days of rhIGF-I treatment (25 xcexcg and 100 xcexcg/kg bid). RhIGF-I also improved insulin sensitivity and glycemic control in some patients with severe type A insulin resistance (Schoenle el al., Diabetologia, 34: 675-679 (1991); Morrow el al., Diabetes, 42 (Suppl.): 269 (1993) (abstract); Kuzuya et al., Diabetes, 42: 696-705 (1993)) and in other patients with non-insulin dependent diabetes mellitus (Schalch et al., xe2x80x9cShort-term metabolic effects of recombinant human insulin-like growth factor I (rhIGF-I) in type II diabetes mellitusxe2x80x9d, in: Spencer E M, ed., Modern Concepts of Insulin-like Growth Factors (New York: Elsevier 1991) pp. 705-713, Zenobi et al., J. Clin. Invest., 90: 2234-2241 (1992)).
A general scheme for the etiology of some clinical phenotypes that give rise to insulin reseistance and the possible effects of administration of IGF-I on selected representative subjects is given in several references. See, e.g., Elahi et as., xe2x80x9cHemodynamic and metabolic responses to human insulin-like growth factor-I (IGF-I) in men,xe2x80x9d in: Modern Concepts of Insulin-Like Growth Factors, (Spencer, E M, ed.), Elsevier, N.Y., pp. 219-224 (1991); Quin et a., New Engl. J. Med., 323: 1425-1426 (1990); Schalch et al., xe2x80x9cShort-term metabolic effects of recombinant human insulin-like growth factor 1 (rhIGF-I) in type II diabetes mellitus,xe2x80x9d in: Modern Concepts of Insulin-Like Growth Factors, (Spencer, E M, ed.), Elsevier, N.Y., pp. 705-713 (1991); Schoenle et al., Diabetologia, 34: 675-679 (1991); Usala et al., N. Eng. J. Med., 327: 853-857 (1992); Lieberman et al., J. Clin. Endo. Metab., 75: 30-36(1992); Zenobi et al., J. Clin. Invest., 90: 2234-2241 (1992); Zenobi et al., J. Clin. Invest., 89: 1908-1913 (1992); Kerr et al., J. Clin. Invest., 91: 141-147 (1993): When IGF-I was used to treat Type II diabetic patients in the clinic at a dose of 120-160 xcexcg/kg twice daily, the side effects outweighed the benefit of the treatment (Jabri et al., Diabetes, 43: 369-374 (1994)). See also Wilton, Acta Paediatr., 383: 137-141 (1992) regarding side effects observed upon treatment of patients with IGF-I.
The IGF binding proteins (IGFBPs) are a family of at least six proteins (Jones and Clemmons, Endocr. Rev., 16: 3-34 (1995); Bach and Rechler, Diabetes Reviews, 3: 38-61 (1995)), with other related proteins also possibly binding the IGFs. The IGFBPs bind IGF-I and IGF-II with varying affinities and specificities (Jones and Clemmons, supra; Bach and Rechler, supra). For example, IGFBP-3 binds IGF-I and IGF-II with a similar affinity, whereas IGFBP-2 and IGFBP-6 bind IGF-II with a much higher affinity than athey bind IGF-I (Bach and Rechler, supra; Oh et al., Endocrinology, 132, 1337-1344 (1993)).
Unlike most other growth factors, the IGFs are present in high concentrations in the circulation, but only a small fraction of the IGFs is not protein bound. For example, it is generally known that in humans or rodents, less than 1% of the IGFs in blood is in a xe2x80x9cfreexe2x80x9d or unbound form (Juul et al., Clin. Endocrinol., 44: 515-523 (1996); Hizuka et al., Growth Regulation, 1: 51-55 (1991); Hasegawa et al., J. Clin. Endocrinol. Metab., 80: 3284-3286 (1995)). The overwhelming majority of the IGFs in blood circulate as part of a non-covalently associated ternary complex composed of IGF-I or IGF-II, IGFBP-3, and a large protein termed the acid-labile subunit (ALS) This complex is composed of equimolar amounts of each of the three components. The ternary complex of an IGF, IGFBP-3, and ALS has a molecular weight of approximately 150,000 daltons, and it has been suggested that the function of this complex in the circulation may be to serve as a reservoir and buffer for IGF-I and IGF-II, preventing rapid changes in free IGF-I or I GF-II.
IGF-I naturally occurs in human body fluids, for example, blood and human cerebral spinal fluid. Although IGF-I is produced in many tissues, most circulating IGF-I is believed to be synthesized in the liver. The IGFBPs are believed to modulate the biological activity of IGF-I (Jones and Clemmons, supra), with IGFBP-1 (Lee et al., Proc. Soc. Exp. Biol. and Med., 204: 4-29 (1993)) being implicated as the primary binding protein involved in glucose metabolism (Baxter, xe2x80x9cPhysiological roles of IGF binding cl proteinsxe2x80x9d, in: Spencer (Ed.), Modern Concepts of Insulin-like Growth Factors (Elsevier, N.Y., 1991), pp. 371-380). IGFBP-1 production by the liver is regulated by nutritional status, with insulin directly suppressing its production (Suikkari et al., J. Clin. Endocrinol. Metab., 66: 266-272 (1988)).
The function of IGFBP-1 in vivo is poorly understood. The administration of purified human IGFBP-1 to rats has been shown to cause an acute, but small, increase in blood glucose (Lewitt et al., Endocrinology, 129: 2254-2256 (1991)). The regulation of IGFBP-1 is somewhat better understood. It has been proposed (Lewitt and Baxter, Mol. Cell Endocrinology, 79: 147-152 (1991)) that when blood glucose rises and insulin is secreted, IGFBP-1 is suppressed, allowing a slow increase in xe2x80x9cfreexe2x80x9d IGF-I levels that might assist insulin action on glucose transport. Such a scenario places the function of IGFBP-1 as a direct regulator of blood glucose.
The IGF system is also composed of membrane-bound receptors for IGF-I, IGF-II, and insulin. The Type 1 IGF receptor is closely related to the insulin receptor in structure and shares some of its signaling pathways (Jones and Clemmons, supra). The IGF-II receptor is a clearance receptor that appears not to transmit an intracellular signal (Jones and Clemmons, supra). Since IGF-I and IGF-II bind to the Type 1 IGF-I receptor with a much higher affinity than to the insulin receptor, it is most likely that most of the effects of IGF-I and IGF-II are mediated by the Type 1 IGF receptor (Ballard et al., xe2x80x9cDoes IGF-I ever act through the insulin receptor?xe2x80x9d, in Baxter et al. (Eds.), The Insulin-Like Growth Factors and Their Regulatory Proteins, (Amsterdam: Elsevier, 1994), pp. 131-138).
There has been much work identifying the domains on IGF-I and IGF-II that bind to the IGFBPs (Bayne et al., J. Biol. Chem., 265: 15648-15652 (1990); Dubaquie and Lowman, Biochemistry, 38: 6386-6396 (1999); U.S. Pat. Nos. 5,077,276; 5,164,370; 5,470,828). For example, it has been discovered that the N-terminal region of IGF-I and IGF-II is critical for binding to the IGFBPs (U.S. Pat. Nos. 5,077,276; 5,164,370; 5,470,828). Thus, the natural IGF-I variant, designated des(1-3)IGF-I, binds poorly to IGFBPs.
A similar amount of research has been devoted to identifying the domains on IGF-I and IGF-II that bind to the Type 1 IGF receptor (Bayne et al., supra; Oh et al., supra). It was found that the tyrosine residues in IGF-I at positions 24, 31, and 60 are crucial to the binding of IGF-I to the Type 1 IGF receptor (Bayne et al., supra). Mutant IGF-I molecules where one or more of these tyrosine residues are substituted showed progressively reduced binding to Type 1 IGF receptors. Bayne et al., supra, also investigated whether such mutants of IGF-I could bind to the Type 1 IGF receptor and to the IGFBPs. They found that quite different residues on IGF-I and IGF-II are used to bind to the IGFBPs from those used to bind to the Type 1 IGF receptor. It is therefore possible to produce IGF variants that show reduced binding to the IGFBPs, but, because they bind well to the Type 1 IGF receptor, show maintained activity in in vitro activity assays.
Also reported was an IGF variant that binds to IGFBPs but not to IGF receptors and therefore shows reduced activity in in vitro activity assays (Bar et al., Endocrinology, 127: 3243-3245 (1990)). In this variant, designated (1-27,gly4,38-70)-hIGF-I, residues 28-37 of the C region of human IGF-I are replaced by a four-residue glycine bridge. Bar et al. studied the transport of the mutant IGF-I when it was perfused as a complex with IGFBP through the heart in terms of the localization of IGFBPs bound to the mutant IGF or to IGF itself. There were no data supplied by Bar et al. on the localization of the IGF mutant given alone, only data on the localization of the complex of the IGF mutant and IGFBP. Further, Bar et al. provided no data on any biological or efficacy response to the administration of the IGF mutant.
Other truncated IGF-I variants are disclosed. For example, in the patent literature, WO 96/33216 describes a truncated variant having residues 1-69 of authentic IGF-I. EP 742,228 discloses two-chain IGF-I superagonists which are derivatives of the naturally occurring single-chain IGF-I having an abbreviated C domain. The IGF-I analogs are of the formula: BCn,A wherein B is the B domain of IGF-I or a functional analog thereof, C is the C domain of IGF-I or a functional analog thereof, n is the number of amino acids in the C domain and is from about 6 to about 12, and A is the A domain of IGF-I or a functional analog thereof.
Additionally, Cascieri et al., Biochemistry, 27: 3229-3233 (1988) discloses four mutants of IGF-I, three of which have reduced affinity to the Type 1 IGF receptor. These mutants are: (Phe23,Phe24,Tyr25)IGF-I (which is equipotent to human IGF-I in its affinity to the Types 1 and 2 IGF and insulin receptors), (Leu24) IGF-I and (Ser24) IGF-I (which have a lower affinity than IGF-I to the human placental Type 1 IGF receptor, the placental insulin receptor, and the Type 1 IGF receptor of rat and mouse cells), and desoctapeptide (Leu24)IGF-I (in which the loss of aromaticity at position 24 is combined with the deletion of the carboxyl-terminal D region of hIGF-I, which has lower affinity than (Leu24)IGF-I for the Type 1 receptor and higher affinity for the insulin receptor). These four mutants have normal affinities for human serum binding proteins.
Bayne et al., J. Biol. Chem., 263: 6233-6239 (1988) discloses four structural analogs of human IGF-I: a B-chain mutant in which the first 16 amino acids of IGF-I were replaced with the first 17 amino acids of the B-chain of insulin, (Gln3,Ala4) IGF-I, (Tyr15,Leu16)IGF-I, and (Gln3Ala4,Tyr15,Leu16) IGF-I. These studies identify some of the domains of IGF-I that are responsible for maintaining high-affinity binding with the serum binding protein and the Type 2 IGF receptor.
Bayne et al., J. Biol. Chem., 264: 11004-11008 (1988) discloses three structural analogs of IGF-I: (1-62)IGF-I, which lacks the carboxyl-terminal 8-amino-acid D region of IGF-I; (1-27,Gly4,38-70)IGF-I, in which residues 28-37 of the C region of IGF-I are replaced by a four-residue glycine bridge; and (1-27,Gly4,38-62)IGF-I, with a C region glycine replacement and a D region deletion. Peterkofsky et al., Endocrinology, 128: 1769-1779 (1991) discloses data using the Gly4 mutant of Bayne et al., supra (Vol. 264). U.S. Pat No. 5,714,460 refers to using IGF-I or a compound that increases the active concentration of IGF-I to treat neural damage.
Cascieri et al., J. Biol. Chem., 264: 2199-2202 (1989) discloses three IGF-I analogs in which specific residues in the A region of IGF-I are replaced with the corresponding residues in the A chain of insulin. The analogs are: (Ile41,Glu45,Gln46,Thr49,Ser50,Ile50,Ser53,Tyr55,Gln56)IGF-I, an A chain mutant in which residue 41 is changed from threonine to isoleucine and residues 42-56 of the A region are replaced; (Thr49,Ser50,Ile51)IGF-I; and (Tyr55,Gln56)IGF-I.
Clemmons et al., J. Biol. Chem., 265: 12210-12216 (1990) discloses use of IGF-I analogs that have reduced binding affinity for either the Type 1 IGF receptor or binding proteins to study the ligand specificity of IGFBP-1 and the role of IGFBP-1 in modulating the biological activity of IGF-I.
WO 94/04569 discloses a specific binding molecule, other than a natural IGFBP, that is capable of binding to IGF-I and can enhance the biological activity of IGF-I.
U.S. Pat. Nos. 5,593,844 and 5,210,017 disclose a ligand-mediated immunofunctional binding protein assay method that can be used to quantitate the amount of GH binding protein or IGFBP in a liquid sample by the use of antibodies, where complex formation takes place between one of these binding proteins and the hormone ligand that binds to it.
The direction of research into IGF variants has mostly been is to make IGF variants that do not bind to the IGFBPs but show maintained binding to the IGF receptor. The idea behind the study of such molecules is that the major actions of the IGFBPs are proposed to be an inhibition of the activity of the IGFs. Chief among these variants is the natural molecule, des(1-3)IGF-I, which shows selectively reduced affinity for some of the IGF binding proteins, yet a maintained affinity for the IGF receptor (U.S. Pat. Nos. 5,077,276; 5,164,370; 5,470,828, supra).
Peptides which bind to IGFBP-1, block IGF-I binding to this binding protein, and thereby release xe2x80x9cfree-IGFxe2x80x9d activity from mixtures of IGF-I and IGFBP-1 have been recently described (Lowman et al., Biochemistry, 37: 8870-8878 (1998); WO 98/45427 published Oct. 15, 1998; Lowman et al., International Pediatric Nephrology Association, Fifth Symposium on Growth and Development in Children with Chronic Renal Failure (New York, Mar. 13, 1999)). These include bp1-02, a cyclic (disulfide-containing) peptide discovered from phage-displayed peptide libraries, as well as truncated forms of this peptide: bp1-01 and bp1-16 (WO 98/45427, supra). Peptide inhibition assays showed that bp1-01 and bp1-02 inhibited IGFBP-1 binding to IGF-I with IC50 values of 180 nM and 50 nM, respectively; and cell-based assays showed release of xe2x80x9cfree-IGFxe2x80x9d activity with EC50 values of 400 nM and 190 nM, respectively (Lowman et al., supra, 1998).
There is a need in the art for a molecule that acts as an IGF agonist, and also for a molecule that binds to IGF binding proteins with high affinity and specificity for therapeutic or diagnostic purposes.
Additional structure-function studies and affinity maturation beyond that disclosed in Lowman et al., supra, 1998 and WO 98/45427, supra, have been performed using further natural and non-natural amino acid substitutions as well as multiple (combined) substitutions in peptide variants of the bp1-01 family. Unless otherwise specified, all peptides described here are cyclic, containing disulfides between bp1-01 residues Cys-1 and Cys-10.
Accordingly, the present invention relates, in a first embodiment, to a peptide comprising the following sequence:
Xaa(1-4)CysXaa(6)Xaa(7)GlyXaa(9)Xaa(10)Xaa(11)Xaa(12)Xaa(13)CysXaa(15)Xaa(16)Xaa(17) Xaa(18) (SEQ ID NO:1), wherein Xaa(1-4) is absent or is between 1 and 4 amino acids of any kind, Xaa(6), Xaa(7), Xaa(9), Xaa(11), Xaa(15), and Xaa(16) are independently any amino acid, Xaa(10) and Xaa(11) are independently Leu or Nle, and Xaa(12), Xaa(17), and Xaa(18) are independently Nal(1), His, Phe, Trp, Tyr, Pro, Gln, or Met.
In one preferred embodiment, this peptide comprises the following sequence:
GluAlaArgValCysArgAlaGlyProLeuGlnTrpLeuCysGluLysTyrPhe (SEQ ID NO:2).
In another preferred embodiment, this peptide comprises the following sequence:
CysXaa(6)Xaa(7)GlyXaa(9)Xaa(10)Xaa(11)TrpXaa(13)CysXaa(15)Xaa(16)Xaa(17)Xaa(18) (SEQ ID NO:3).
More preferably, such peptide comprises one of the following sequences:
CysArgAlaGlyAlaLeuGlnTrpLeuCysGluLysTyrPhe (SEQ ID NO:4);
CysArgAlaGlyArgLeuGlnTrpLeuCysGluLysTyrPhe (SEQ ID NO:5);
CysArgAlaGlyAsnLeuGlnTrpLeuCysGluLysTyrPhe (SEQ ID NO:6);
CysArgAlaGlyProNleGlnTrpLeuCysGluLysTyrPhe (SEQ ID NO:7);
CysArgAlaGlyProLeuGlnTrpNleCysGluLysTyrPhe (SEQ ID NO:8);
CysArgAlaGlyProLeuGlnArgLeuCysGluLysTyrPhe (SEQ ID NO:9);
CysArgAlaGlyProLeuGlnNal(1)LeuCysGluLysTyrPhe (SEQ ID NO:10); or
CysArgAlaGlyProLeuGlnHisLeuCysGluLysTyrPhe (SEQ ID NO:11).
In another preferred embodiment of SEQ ID NO:1, C-terminal to the C-terminal Xaa(18)(IL) is the sequence Xaa(19)ThrTyr, wherein Xaal(19) is any amino acid. More preferred such peptides comprise the following sequence: Xaa(1-4)CysArgAlaGlyProLeuGlnTrpLeuCysGluXaa(16)TyrPheXaa(19)ThrTyr (SEQ ID NO:12), wherein Xaa(16) is Lys or His and Xaa(19) is Ala, Ser, Gln, Asp, Glu, or Lys. More preferably, such peptides comprise one of the following sequences:
SerGluValGlyCysArgAlaGlyProLeuGlnTrpLeuCysGluLysTyrPheSerThrTyr (SEQ ID NO:13);
SerGluValGlyCysArgAlaGlyProLeuGlnTrpLeuCysGluLysTyrPheAlaThrTyr (SEQ ID NO:14);
SerGluValGlyCysArgAlaGlyProLeuGlnTrpLeuCysGluLysTyrPheGlnThrTyr (SEQ ID NO:15);
SerGluValGlyCysArgAlaGlyProLeuGlnTrpLeuCysGluLysTyrPheGlnThrThr (SEQ ID NO:16);
SerGluValGlyCysArgAlaGlyProLeuGlnTrpLeuCysGluLysTyrPheAspThrTyr (SEQ ID NO:17);
SerGluValGlyCysArgAlaGlyProLeuGlnTrpLeuCysGluLysTyrPheGluThrTyr (SEQ ID NO:18);
SerGluValGlyCysArgAlaGlyProLeuGlnTrpLeuCysGluLysTyrPheLysThrTyr (SEQ ID NO:19);
GluAlaArgValCysArgAlaGlyProLeuGlnTrpLeuCysGluLysTyrPheSerThrTyr (SEQ ID NO:20);
GlyGlnGlnSerCysArgAlaGlyProLeuGlnTrpLeuCysGluLysTyrPheSerThrTyr (SEQ ID NO:21);
AlaSerSerMetCysArgAlaGlyProLeuGlnTrpLeuCysGluLysTyrPheSerThrTyr (SEQ ID NO:22);
GlnGlyProAspCysArgAlaGlyProLeuGlnTrpLeuCysGluLysTyrPheSerThrTyr (SEQ ID NO:23);
GlnAlaSerGluCysArgAlaGlyProLeuGlnTrpLeuCysGluLysTyrPheSerThrTyr (SEQ ID NO:24);
AlaGluThrLeuCysArgAlaGlyProLeuGlnTrpLeuCysGluLysTyrPheSerThrTyr (SEQ ID NO:25);
AsnSerLeuLeuCysArgAlaGlyProLeuGlnTrpLeuCysGluLysTyrPheSerThrTyr (SEQ ID NO:26);
AlaGlnTrpValCysArgAlaGlyProLeuGlnTrpLeuCysGluLysTyrPheSerThrTyr (SEQ ID NO:27);
GlyGlnGlnSerCysAlaAlaGlyProLeuGlnTrpLeuCysGluHisTyrPheSerThrTyr (SEQ ID NO:28); or
GlyGlnGlnSerCysAlaAlaGlyProLeuGlnTrpLeuCysGluHisTyrPheSerThrTyrGlyArg (SEQ ID NO:29).
In another specific embodiment, the invention relates to a peptide comprising SEQ ID NO:4; SEQ ID NO:5; SEQ ID NO:6; SEQ ID NO:7;
SEQ ID NO:8; SEQ ID NO:9; SEQ ID NO:10; SEQ ID NO:11; SEQ ID NO: 14;
SEQ ID NO:15; SEQ ID NO:16; SEQ ID NO:17; SEQ ID NO:18; SEQ ID NO:19;
SEQ ID NO:20; SEQ ID NO:21; SEQ ID NO:22; SEQ ID NO:23; SEQ ID NO:24; SEQ ID NO:25; SEQ ID NO:26; SEQ ID NO:27; SEQ ID NO:28; or SEQ ID NO:29.
In another aspect of the invention, the above peptide having SEQ ID NO:1 or SEQ ID NO:3 has a C-terminal fusion comprising the following sequence:
GlyGlyGlySerGlyGlyAlaGlnHisAspGluAlaValAspAsnLysPheAsnLysGluGlnGlnAsnAlaPheTyrGluIleLeuHisLeuProAsnLeuAsnGluGluGlnArgAsnAlaPheIleGlnSerL euLysAspAspProSerGlnSerAlaAsnLeuLeuAlaGluAlaLysLysLeuAsnAspAlaGlnAlaProAsnValAspMetAsn (SEQ ID NO:30).
In another embodiment, this invention relates to a constrained helical peptide comprising a sequence of nine amino acid residues having a first terminal residue and a second terminal residue, wherein said residues flank an internal sequence of seven amino acids and have side-chains covalently bonded to each other to form a locking moiety and thereby constrain the peptide. Preferably, the internal sequence is Xaa(7)LeuAlaXaa(10)Xaa(11)Xaa(12)Xaa(13) (SEQ ID NO:31), wherein Xaa(7), Xaa(11), Xaa(12), and Xaa(13) are independently Nal(1), His, Phe, Trp, Tyr, Pro, Gln, or Met, and Xaa(10) is any amino acid.
In yet another embodiment, the invention relates to a peptide comprising the following sequence:
Xaa(1-4)Xaa(5), Xaa(6-7)ProLeuGluXaa(11)LeuAlaXaa(14)Xaa(15)Xaa(16)Xaa(17)GluXaa(19) (SEQ ID NO:32), wherein Xaa(1-4) is absent or is between 1 and 4 amino acids of any kind; Xaa(5) is any amino acid, Xaa(6-7) is absent or is between 1 and 2 amino acids, Xaa(14) and Xaa(15) are independently any amino acid, Xaa(11) and Xaa(16) are independently Nal(1), His, Phe, Trp, Tyr, Pro, Gln, or Met, Xaa(17) is absent or is Nal(1), His, Phe, Trp, Tyr, Pro, Gln, or Met, and Xaa(19) is absent or is Gly.
In a preferred peptide of this type, C-terminal to the C-terminal Xaa(19) is the sequence Xaa(20)ThrTyr, wherein Xaa(20) is any amino acid.
More specifically, the peptide comprises the following sequence:
Xaa(5)Xaa(6-7)ProLeuGluXaa(11)LeuAlaXaa(14)Xaa(15)Xaa(16)Xaa(17)GluGly (SEQ ID NO:33), wherein Xaa(6-7) is two amino acids. Other preferred peptides of this type comprise one of the following sequences:
ArgAlaGlyProLeuGluTrpLeuAlaGluLysTyrGluGly (SEQ ID NO:34);
ArgProLeuGluTrpLeuAlaGluLysTyrPheGlu (SEQ ID NO:35); or
ArgAlaGlyProLeuGluTrpLeuAlaGluLysTyrPheGlu (SEQ ID NO:36).
Any of the above peptides preferably contains 10-60 amino acids, more preferably 12-25 amino acids.
Also provided herein is a composition comprising one of the peptides described above in a carrier. Preferably, this composition is sterile and the carrier is a pharmaceutically acceptable carrier.
Uses of these peptides include all uses that liberate or enhance at least one biological activity of exogenous or endogenous IGFs. They can be used in treating, inhibiting, or preventing conditions in which an IGF such as IGF-I is useful, as described below.
The invention also provides a method of constructing a constrained helical peptide, comprising the steps of:
(a) synthesizing a peptide comprising a sequence of nine amino acid residues having a first terminal residue and a second terminal residue that flank an internal sequence of seven amino acid residues and have side-chains containing an amide bond-forming substituent;
(b) providing a difunctional linker having a first functional group capable of forming an amide linkage with the side-chain amide bond-forming substituent of the first terminal residue and having a second functional group capable of forming an amide linkage with the side-chain amide bond-forming substituent of the second terminal residue; and
(c) cyclizing the peptide by reacting the side-chain amide bond-forming substituent of the first terminal residue with the first functional group of the difunctional linker to form an amide linkage and reacting the side-chain amide bond-forming substituent of the second terminal residue with the second functional group of the difunctional linker to form an amide linkage, yielding a constrained helical peptide.
In a preferred embodiment, in step (a) the side-chain amide bond-forming substituent of the first terminal residue is protected with a first protecting group and the side-chain amide bond-forming substituent of the second terminal residue is protected with a second protecting group, wherein the first protecting group and the second protecting group are differentially removable, and wherein in step (c) the first protecting group is removed such that the side-chain amide bond-forming substituent of the first terminal residue is deprotected and the side-chain amide bond-forming substituent of the second terminal residue is not deprotected before the peptide is reacted with the difunctional linker, and thereafter the peptide is reacted with the difunctional linker to form an amide linkage between the side-chain amide bond-forming substituent of the first terminal residue and the first functional group of the difunctional linker, and thereafter the second protecting group is removed from the side-chain amide bond-forming substituent of the second terminal residue and the peptide is cyclized by intramolecularly reacting the side-chain amide bond-forming substituent of the second terminal residue with the second functional group of the difunctional linker to form an amide linkage.
In another aspect, the invention provides a method of constructing a constrained helical peptide, comprising the steps of:
(a) synthesizing a peptide comprising a sequence of nine amino acid residues having a first terminal residue and a second terminal residue that flank an internal sequence of seven amino acid residues and have a side-chain containing an amide bond-forming substituent, wherein the first terminal residue is coupled to a difunctional linker having a first functional group and a second functional group, wherein the first functional group is in an amide linkage with the side-chain amide bond-forming substituent of the first terminal residue, and wherein the second functional group of the difunctional linker is capable of forming an amide linkage with the side-chain amide bond-forming substituent of the second terminal residue; and
(b) cyclizing the peptide by intramolecularly reacting the side-chain amide bond-forming substituent of the second terminal residue with the second functional group of the difunctional linker to form an amide linkage and thereby yield a constrained helical peptide.
In another embodiment, the invention provides a constrained helical peptide made according to one of the above methods.
Additionally provided herein is a method for increasing serum and tissue levels of biologically-active IGF in a mammal comprising administering to the mammal an effective amount of any of the above peptides. The mammal is preferably human. Also preferred is where administering the peptide, preferably in an amount effective to produce body weight gain, causes an increase in anabolism in the mammal. Additionally preferred is that glycemic control is effected in the mammal after the peptide is administered.
Any of the peptides herein can be administered alone or rn together with another agent such as GH, a GH releasing peptide (GHRP), a GH releasing factor (GHRF), a GH releasing hormone (GHRH), a GH secretagogue, an IGF, an IGF in combination with an IGFBP, an IGFBP, GH in combination with a GH binding protein (GHBP), insulin, or a hypoglycemic agent (which includes in the definition below an insulin-sensitizing agent such as thiazolidinedione).
In another embodiment, a method is provided for determining appropriate dosing of one of the above peptides comprising:
(a) measuring the level of an IGF in a body fluid;
(b) contacting the fluid with a peptide herein using single or multiple doses; and
(c) re-measuring the level of an IGF in the fluid, wherein if the fluid IGF level has fallen by an amount sufficient to produce the desired efficacy for which the peptide is to be administered, then the dose of the peptide is adjustable or adjusted to produce maximal efficacy.
In yet another embodiment, a method is provided for determining the amount of a particular IGFBP or the amount of one of the above peptides bound to a particular IGFBP in a biological fluid so that dosing of the peptide can be adjusted appropriately. This method involves:
(a) contacting the fluid with 1) one of the above-identified peptides and 2) a first antibody attached to a solid-phase carrier, wherein the first antibody is specific for epitopes on the IGFBP such that in the presence of antibody the IGF binding sites remain available on the IGFBP for binding to the peptide, thereby forming a complex between the first antibody and the IGFBP, for a period of time sufficient to saturate all available IGF binding sites on the IGFBP, thereby forming a saturated complex;
(b) contacting the saturated complex with a detectably labeled second antibody which is specific for epitopes on the peptide which are available for binding when the peptide is bound to the IGFBP; and
(c) quantitatively analyzing the amount of the labeled second antibody bound as a measure of the IGFBP in the biological fluid, and therefore as a measure of the amount of the peptide bound.
Also contemplated herein is a kit comprising a container containing a pharmaceutical composition containing one of the above peptides and instructions directing the user to utilize the composition. This kit may optionally further comprise a container containing a GH, a GHRP, a GHRF, a GHRH, a GH secretagogue, an IGF, an IGF complexed to an IGFBP, an IGFBP, a GH complexed with a GHBP, insulin, or a hypoglycemic agent.
In another embodiment herein, a method for directing endogenous IGF either away from, or towards, a particular site in a mammal comprising administering to the mammal an effective amount of one of the above peptides herein that is specific for an IGFBP that is either prevalent at, or absent from, the site.
A further embodiment is a method for detecting endogenous or exogenous IGF bound to an IGF binding protein or the amount of any peptide herein or detecting the level of unbound IGF in a biological fluid comprising:
(a) contacting the fluid with 1) a means for detecting the peptide attached to a solid-phase carrier, wherein the means is specific for the peptide such that in the presence of the peptide the IGF binding sites remain available on the peptide for binding to the IGF binding protein, thereby forming a complex between the means and the IGF binding protein; and 2) the peptide for a period of time sufficient to saturate all available IGF binding sites on the IGF binding protein, thereby forming a saturated complex;
(b) contacting the saturated complex with a detectably labeled second means which is specific for the IGF binding protein which are available for binding when the peptide is bound to the IGF binding protein; and
(c) quantitatively analyzing the amount of the labeled means bound as a measure of the IGFBP in the biological fluid, and therefore as a measure of the amount of bound peptide and IGF binding protein, bound IGF and IGF binding protein, or active IGF present in the fluid.
The present invention further provides various dosage forms of any of the peptides of the present invention, including but not limited to, those suitable for parenteral, oral, rectal and pulmonary administration of a peptide. In preferred aspects herein a therapeutic dosage form is provided suitable for inhalation and the invention provides for the therapeutic treatment of diseases or disorders involving an IGF-mediated or associated process or event via pulmonary administration of a peptide of the invention. More particularly, the invention is directed to pulmonary administration of the peptides herein by inhalation. Thus, the present invention provides an aerosol formulation comprising an amount of a peptide of the invention, effective to block or prevent an IGF-mediated or associated process or event and a dispersant. In one embodiment, any one of the above peptides can be provided in a liquid aerosol formulation. Alternatively, the peptide can be provided as a dry powder aerosol formulation. Therefore, according to the present invention, formulations are provided that provide an effective non-invasive alternative to other parenteral routes of administration of the peptides herein for the treatment of IGF-mediated or associated events.
Isolated nucleic acid encoding one of the above peptides herein is also provided, and may be used for in vivo or ex vivo gene therapy.
The peptides herein are superior to IGF mutants such as des(1-3)IGF-I, since the latter have short half-lives and effects, whereas the peptides herein have longer half lives and effects, and this binding avoids normal renal filtration which would otherwise eliminate short peptides and other small molecules rapidly. Further, administering any one of the peptides herein together with exogenous GH or GH secretagogues would have the advantage of minimizing diabetogenic effects of such GH and secretagogues. Yet another advantage of the peptides herein is that there is a ceiling of the effects of the IGF agonist peptide herein. That is, it cannot exert more effects than the maximum capacity of IGFBPs to carry IGFS, unlike IGF-I, which can have unwanted side effects if used in large concentrations over its maximum efficacy.